scholarly journals Localizing a gate in CFTR

2015 ◽  
Vol 112 (8) ◽  
pp. 2461-2466 ◽  
Author(s):  
Xiaolong Gao ◽  
Tzyh-Chang Hwang
Keyword(s):  
Chloride Ions ◽  
Transmembrane Conductance Regulator ◽  
Amino Acid Residues ◽  
Transmembrane Conductance ◽  
Open Probability ◽  
Transmembrane Segments ◽  
Narrow Region ◽  
Ion Translocation ◽  
Transporter Family ◽  
Narrow Section

Experimental and computational studies have painted a picture of the chloride permeation pathway in cystic fibrosis transmembrane conductance regulator (CFTR) as a short narrow tunnel flanked by wider inner and outer vestibules. Although these studies also identified a number of transmembrane segments (TMs) as pore-lining, the exact location of CFTR’s gate(s) remains unknown. Here, using a channel-permeant probe, [Au(CN)2]−, we provide evidence that CFTR bears a gate that coincides with the predicted narrow section of the pore defined as residues 338–341 in TM6. Specifically, cysteines introduced cytoplasmic to the narrow region (i.e., positions 344 in TM6 and 1148 in TM12) can be modified by intracellular [Au(CN)2]− in both open and closed states, corroborating the conclusion that the internal vestibule does not harbor a gate. However, cysteines engineered to positions external to the presumed narrow region (e.g., 334, 335, and 337 in TM6) are all nonreactive toward cytoplasmic [Au(CN)2]− in the absence of ATP, whereas they can be better accessed by extracellular [Au(CN)2]− when the open probability is markedly reduced by introducing a second mutation, G1349D. As [Au(CN)2]− and chloride ions share the same permeation pathway, these results imply a gate is situated between amino acid residues 337 and 344 along TM6, encompassing the very segment that may also serve as the selectivity filter for CFTR. The unique position of a gate in the middle of the ion translocation pathway diverges from those seen in ATP-binding cassette (ABC) transporters and thus distinguishes CFTR from other members of the ABC transporter family.

Mechanism of activation of Xenopus CFTR by stimulation of PKC

2004 ◽  
Vol 287 (5) ◽  
pp. C1256-C1263 ◽  
Author(s):  
Yongyue Chen ◽  
Guillermo A. Altenberg ◽  
Luis Reuss
Keyword(s):  
Single Channel ◽  
Membrane Conductance ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Open Probability ◽  
Substituted Cysteine Accessibility ◽  
Maximal Stimulation ◽  
A Minor ◽  
Cystic Fibrosis Transmembrane ◽  
Stimulation Of

PKA-mediated phosphorylation of the regulatory (R) domain plays a major role in the activation of the human cystic fibrosis transmembrane conductance regulator (hCFTR). In contrast, the effect of PKC-mediated phosphorylation is controversial, smaller than that of PKA, and dependent on the cell type. In the present study, we expressed Xenopus CFTR ( XCFTR) and hCFTR in Xenopus oocytes and examined their responses (i.e., macroscopic membrane conductance) to maximal stimulation by PKC and PKA agonists. With XCFTR, the average response to PKC was approximately sixfold that of PKA stimulation. In contrast, with hCFTR, the response to PKC was ∼90% of the response to PKA stimulation. The reason for these differences was the small response of XCFTR to PKA stimulation. Using the substituted cysteine accessibility method, we found no evidence for insertion of functional CFTR channels in the plasma membrane in response to PKC stimulation. The increase in macroscopic conductance in response to PKC stimulation of XCFTR was due to an approximately fivefold increase in single-channel open probability, with a minor (∼30%) increase in single-channel conductance. The responses of XCFTR to PKC stimulation and of hCFTR to PKA stimulation were mediated by similar increases in Po. In both instances, there were no changes in the number of channels in the membrane. We speculate that in animals other than humans, PKC stimulation may be the dominant mechanism for activation of CFTR.

Evidence for direct CFTR inhibition by CFTRinh-172 based on Arg347 mutagenesis

2008 ◽  
Vol 413 (1) ◽  
pp. 135-142 ◽  
Author(s):  
Emanuela Caci ◽  
Antonella Caputo ◽  
Alexandre Hinzpeter ◽  
Nicole Arous ◽  
Pascale Fanen ◽  
...  
Keyword(s):  
Amino Acids ◽  
Transmembrane Helix ◽  
Protein A ◽  
Transmembrane Conductance Regulator ◽  
Amino Acid Residues ◽  
Transmembrane Conductance ◽  
Inhibitory Potency ◽  
Functional Assays ◽  
Cftr Protein ◽  
Cystic Fibrosis Transmembrane

CFTR (cystic fibrosis transmembrane conductance regulator) is an epithelial Cl− channel inhibited with high affinity and selectivity by the thiazolidinone compound CFTRinh-172. In the present study, we provide evidence that CFTRinh-172 acts directly on the CFTR. We introduced mutations in amino acid residues of the sixth transmembrane helix of the CFTR protein, a domain that has an important role in the formation of the channel pore. Basic and hydrophilic amino acids at positions 334–352 were replaced with alanine residues and the sensitivity to CFTRinh-172 was assessed using functional assays. We found that an arginine-to-alanine change at position 347 reduced the inhibitory potency of CFTRinh-172 by 20–30-fold. Mutagenesis of Arg347 to other amino acids also decreased the inhibitory potency, with aspartate producing near total loss of CFTRinh-172 activity. The results of the present study provide evidence that CFTRinh-172 interacts directly with CFTR, and that Arg347 is important for the interaction.

scholarly journals Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein

1999 ◽  
Vol 277 (4) ◽  
pp. C833-C839 ◽  
Author(s):  
Beate Illek ◽  
Lei Zhang ◽  
Nancy C. Lewis ◽  
Richard B. Moss ◽  
Jian-Yun Dong ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Adrenergic Stimulation ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Nasal Potential Difference ◽  
Cystic Fibrosis Transmembrane

The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and ΔF508) of the cystic fibrosis transmembrane conductance regulator (CFTR). In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was P o = 0.047 ± 0.019. Addition of genistein activated Cl currents ∼10-fold, and the P o of G551D-CFTR increased to 0.49 ± 0.12, which is a P o similar to wild-type CFTR. In cystic fibrosis (CF) epithelial cells homozygous for the trafficking-impaired ΔF508 mutation, forskolin and genistein activated Cl currents only after 4-phenylbutyrate treatment. These data suggested that genistein activated CFTR mutants that were present in the cell membrane. Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, −2.4 ± 0.6 mV, which corresponds to 16.9% of the responses (to β-adrenergic stimulation) found in healthy subjects.

Expression of Drosophila melanogaster P-glycoproteins is associated with ATP channel activity

1996 ◽  
Vol 271 (5) ◽  
pp. C1527-C1538 ◽  
Author(s):  
I. Bosch ◽  
G. R. Jackson ◽  
J. M. Croop ◽  
H. F. Cantiello
Keyword(s):  
Drosophila Melanogaster ◽  
Transmembrane Conductance Regulator ◽  
Channel Activity ◽  
Transmembrane Conductance ◽  
Apparent Permeability ◽  
Transporter Family ◽  
Pathway Expression ◽  
Endogenous Substrate ◽  
Atp Binding Cassettes ◽  
Cystic Fibrosis Transmembrane

Two distinct Drosophila melanogaster P-glycoprotein (Pgp) gene homologues of different chromosomal origin, MDR49 and MDR65, have been previously identified (38). Most Pgps are implicated in the development of the multidrug-resistance phenotype. Despite intense efforts to identify the molecular mechanism(s) associated with Pgp function, the endogenous substrate(s) of these transport molecules is largely unknown. Recent studies from our laboratory indicate that a murine Pgp homologue (E. H. Abraham, A. G. Prat, L. Gerweck, T. Seneveratne, R. J. Arceci, R. Kramer, G. Guidotti, and H. F. Cantiello. Proc. Natl. Acad. Sci. USA 90: 312-316, 1993) and a related protein, the cystic fibrosis transmembrane conductance regulator (CFTR; I. L. Reisin, A. Prat, E. H. Abraham, J. F. Amara, R. J. Gregory, D. A. Ausiello, and H. F. Cantiello. J. Biol. Chem. 269: 20584-20591, 1994), are novel ATP-permeable ion channels. The common feature of these two proteins is the conserved ATP-binding cassettes (ABC); thus molecules structurally linked to the ABC transporter family may be also functionally associated with ATP channel activity. In this study, MDR65 and MDR49 Pgps were functionally expressed in Sf9 cells, and patch-clamp techniques were applied to assess the role of these proteins in the electrodiffusional movement of ATP. In the presence of intracellular ATP and external NaCl, expression of MDR65 was associated with a linear electrodiffusional pathway that was permeable to both ATP and Cl-. Under symmetrical ATP conditions, only voltage depolarization activated a MDR65-mediated ATP-conductive pathway. Expression of MDR49 was also associated with a voltage-activated ATP conductance in symmetrical ATP, but no apparent permeability to either Cl- or ATP was observed under asymmetrical conditions. The different functional properties of MDR65 and MDR49 may be indicative of distinct physiological roles in this organism. The study indicates, however, that the two Drosophila Pgp homologues share strong functional similarities with their mammalian relatives Pgp and CFTR.

scholarly journals Potentiation of the cystic fibrosis transmembrane conductance regulator Cl− channel by ivacaftor is temperature independent

2018 ◽  
Vol 315 (5) ◽  
pp. L846-L857 ◽  
Author(s):  
Yiting Wang ◽  
Zhiwei Cai ◽  
Martin Gosling ◽  
David N. Sheppard
Keyword(s):  
Cystic Fibrosis ◽  
Temperature Dependence ◽  
Single Channel ◽  
Channel Gating ◽  
Transmembrane Conductance Regulator ◽  
Wild Type ◽  
Transmembrane Conductance ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Cystic Fibrosis Transmembrane

Ivacaftor is the first drug to target directly defects in the cystic fibrosis transmembrane conductance regulator (CFTR), which causes cystic fibrosis (CF). To understand better how ivacaftor potentiates CFTR channel gating, here we investigated the effects of temperature on its action. As a control, we studied the benzimidazolone UCCF-853, which potentiates CFTR by a different mechanism. Using the patch-clamp technique and cells expressing recombinant CFTR, we studied the single-channel behavior of wild-type and F508del-CFTR, the most common CF mutation. Raising the temperature of the intracellular solution from 23 to 37°C increased the frequency but reduced the duration of wild-type and F508del-CFTR channel openings. Although the open probability ( Po) of wild-type CFTR increased progressively as temperature was elevated, the relationship between Po and temperature for F508del-CFTR was bell-shaped with a maximum Po at ~30°C. For wild-type CFTR and to a greatly reduced extent F508del-CFTR, the temperature dependence of channel gating was asymmetric with the opening rate demonstrating greater temperature sensitivity than the closing rate. At all temperatures tested, ivacaftor and UCCF-853 potentiated wild-type and F508del-CFTR. Strikingly, ivacaftor but not UCCF-853 abolished the asymmetric temperature dependence of CFTR channel gating. At all temperatures tested, Po values of wild-type CFTR in the presence of ivacaftor were approximately double those of F508del-CFTR, which were equivalent to or greater than those of wild-type CFTR at 37°C in the absence of the drug. We conclude that the principal effect of ivacaftor is to promote channel opening to abolish the temperature dependence of CFTR channel gating.

Potentiation of effect of PKA stimulation of Xenopus CFTR by activation of PKC: role of NBD2

2004 ◽  
Vol 287 (5) ◽  
pp. C1436-C1444 ◽  
Author(s):  
Yongyue Chen ◽  
Brian Button ◽  
Guillermo A. Altenberg ◽  
Luis Reuss
Keyword(s):  
Plasma Membrane ◽  
Xenopus Oocytes ◽  
Single Channel ◽  
Transmembrane Conductance Regulator ◽  
Transmembrane Conductance ◽  
Open Probability ◽  
Substituted Cysteine Accessibility ◽  
Potentiation Effect ◽  
Biophysical Mechanism ◽  
Small Change

Activity of the human (h) cystic fibrosis transmembrane conductance regulator (CFTR) channel is predominantly regulated by PKA-mediated phosphorylation. In contrast, Xenopus ( X)CFTR is more responsive to PKC than PKA stimulation. We investigated the interaction between the two kinases in XCFTR. We expressed XCFTR in Xenopus oocytes and maximally stimulated it with PKA agonists. The magnitude of activation after PKC stimulation was about eightfold that without pretreatment with PKC agonist. hCFTR, expressed in the same system, lacked this response. We name this phenomenon XCFTR-specific PKC potentiation effect. To ascertain its biophysical mechanism, we first tested for XCFTR channel insertion into the plasma membrane by a substituted-cysteine-accessibility method. No insertion was detected during kinase stimulation. Next, we studied single-channel properties and found that the single-channel open probability ( Po) with PKA stimulation subsequent to PKC stimulation was 2.8-fold that observed in the absence of PKC preactivation and that single-channel conductance (γ) was increased by ∼22%. To ascertain which XCFTR regions are responsible for the potentiation, we constructed several XCFTR-hCFTR chimeras, expressed them in Xenopus oocytes, and tested them electrophysiologically. Two chimeras [hCFTR NH2-terminal region or regulatory (R) domain in XCFTR] showed a significant decrease in potentiation. In the chimera in which XCFTR nucleotide-binding domain (NBD)2 was replaced with the hCFTR sequence there was no potentiation whatsoever. The converse chimera (hCFTR with Xenopus NBD2) did not exhibit potentiation. These results indicate that potentiation by PKC involves a large increase in Po (with a small change in γ) without CFTR channel insertion into the plasma membrane, that XCFTR NBD2 is necessary but not sufficient for the effect, and that the potentiation effect is likely to involve other CFTR domains.

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